Difference between revisions of "Part:BBa J10050"

Revision as of 17:00, 21 October 2019

weak promoter + weak RBS + lacZa.GFP fusion

Designed and built by Natalie Kuldell for BioBuilder teacher's kit. Weak constitutive promoter, weak RBS and the lacZa.GFP fusion protein.

Sequence and Features

Usage and Biology

Lambert_GA 2019 Characterization

Lambert_GA 2019 tested several combinations of constitutive promoters and ribosomal binding sites to characterize each by measuring enzyme activity and therefore protein expression. The gene expressed, LacZ, codes for β-galactosidase (β-gal), which typically breaks down lactose. Instead of using lactose, we added the sugar ONPG (Ortho-Nitrophenyl-β-galactoside). β-gal breaks ONPG down into galactose and ONP (Ortho-Nitrophenol), which has a yellow color. If there is more ONP present, there is more enzymatic activity and therefore more expression of LacZ. We used a plate reader to measure absorbance at 420 nm, measuring yellow color, and 600nm, measuring cell density. We inputted those absorbance values into the Miller unit formula to calculate enzymatic activity per cell per milliliter.